Device for examining chemical and/or biological samples

ABSTRACT

An apparatus for examination of chemical and/or biological samples, comprises a sample support ( 14 ) for receiving the samples ( 16 ). Further, the apparatus comprises an objective ( 10 ) for observation of the sample ( 16 ) through a sample support wall ( 18 ). A gap ( 22 ) is formed between an outer surface ( 20 ) of the sample support wall ( 18 ) and an exit lens ( 12 ) of the objective ( 10 ). According to the invention, an automatic supply device ( 24 ) for automatic supply of immersion medium ( 46 ) is provided between the outer surface ( 20 ) of the sample support wall ( 18 ) and the exit lens ( 12 ) of the objective ( 10 ).

[0001] The invention relates to an apparatus for examination of chemicaland/or biological samples by means of optical devices. Such an apparatuscan be provided e.g. as a microscope, particularly a confocalmicroscope.

[0002] Apparatus for examination of the above type comprise an objectivefor observation of the sample. When the objective is used, for instance,to observe a sample arranged in a sample support from below through asample carrier bottom which is transmissive to the respectiveobservation radiation, the given constellation of the refractive indiceswill have an unfavorable effect on the course of the path of rays,particularly in case of high numerical objectives. Varying refractiveindices occur in the transition regions between is the exit lens of theobjective and the ambient air, as well as between the bottom of thesample support and the medium arranged between the objective and thesample carrier bottom.

[0003] Particularly in confocal microscopes which are used in highthroughput screening, the focus has to be very small. This is requiredsince, in high throughput screening, examination is performed on sampleshaving low volumes in the μl range or below. Since the amount of theradiation emitted by the sample and taken in by the objective(collection efficiency) has a considerable influence on the measurementtime, the aperture of the objective has to be as high as possible. Thisis of importance particularly in high throughput screening because themeasurement time is one of the decisive parameters.

[0004] Normally, by use of a pipette or the like, the immersion liquidis applied manually to the exit lens of the objective with a refractiveindex >1. Particularly in high throughput screening systems and fullyautomated microscopes and the like, this is extremely cumbersome becausethe objective is arranged at a small distance to a sample support suchas a titration plate.

[0005] For applying the immersion liquid, the objective has to beretracted downwards so that the user can apply immersion liquid onto theexit lens by means of a pipette or the like. Subsequently, the objectiveis advanced to the sample support again until the gap between the exitlens of the objective and the outer surface of the sample support hasbeen filled with the immersion medium. Alternatively, the objective canbe stationary and the sample support can be moved.

[0006] A further disadvantage resides in that, when examining samples intitration plates, i.e. in sample supports comprising a plurality ofindividual samples, a relative movement will occur between the objectiveand the titration plate because the objective has to be advanced to eachindividual sample or vice is versa. In this relative movement, part ofthe immersion liquid will always remain on the underside of the samplesupport so that, after examination of several samples, the gap betweenthe exit lens of the objective and the outer surface of the samplesupport may not be filled completely with immersion liquid anymore.

[0007] It is an object of the invention to facilitate the provision ofimmersion liquid in optical examination apparatus.

[0008] According to the invention, the above object is achieved by anapparatus according to claim 1 and a method according to claim 16,respectively.

[0009] According to the invention, the proposed apparatus forexamination of chemical and/or biological samples is provided with asupply device for automatic supply of immersion medium. By means of thesupply device, immersion medium can be automatically supplied between asample support serving for receiving the samples, and an exit lens of anobjective provided for observation of the samples. Thus, during theobservation of a sample, the gap between the exit lens and the objectivecan always be filled with immersion medium. Instead of a sample support,a means for receiving or holding the sample support can be provided. Inthe given case, the immersion medium will then not touch the samplesupport itself but a plate of the holder or the like.

[0010] Since, according to the invention, the supply of the immersionmedium by means of the supply device is carried out automatically, nouser intervention is required in the apparatus, particularly in the highthroughput screening system or another automatic device. Instead, thesupply device can be configured to the effect that also a retracting ofthe objective, i.e. a removal of the objective from the outer surface ofa sample support wall of the sample support or a movement of the samplesupport will not be required. The inventive apparatus makes it possibleto supply an immersion medium in the measurement position of theobjective, i.e. in that position in which the objective is arranged alsoduring the examination of the sample.

[0011] The inventive supply of immersion medium is preferably performedin that a thin film of immersion medium is formed between the objectiveand the outer surface of the sample support wall. It is particularlypreferred that the width of the gap is selected, in dependence on theimmersion medium used, in such a manner that the immersion medium iscaused to enter the gap under the effect of capillary forces.

[0012] For this purpose, the supply device preferably comprises a supplytube connected to the objective. In this embodiment, the supply tube isheld by the objective so that an exit opening of the supply tube willalways maintain a defined position relative to the exit lens of theobjective. Preferably, the supply tube is arranged in a manner allowingfor the realization of a rigid connection of the supply tube with theobjective. The exit opening of the supply tube is thus arranged outsidethe relevant path of rays for the measurement so that the supply tubedoes not cast shadows.

[0013] The exit opening of the supply tube is arranged as close aspossible to the exit lens. Preferably, the distance is smaller than 15mm, particularly smaller than 10 mm.

[0014] It is preferred that the cross sectional area of the exitopening, which preferably is flattened, is smaller than 1 mm²,particularly smaller than 0.8 mm².

[0015] To guarantee a most uniform and bubble-free application of theimmersion medium onto the exit lens of the objective, the exit openingof the supply tube is preferably arranged at a displacement relative toa central point of the exit lens in the direction of the objective. In aconfiguration where the objective is oriented upwards, this means thatthe exit opening of the supply tube is located below a maximal point ofthe exit lens. Thereby, it is further safeguarded that the supply tubedoes not touch the sample support.

[0016] For conveying the immersion medium, the supply device comprises apump, for instance. The preferably controllable pump will preferably bea controllable hose pump arranged to act on a hose connected to thesupply tube. The provision of a hose pump offers the advantage that noparts of the pump, particularly no metallic parts, will get into contactwith the immersion medium so that the immersion medium will not beaffected by the pump.

[0017] A further possibility for the supply of the immersion mediumresides in the provision of a supply container which is connected to thesupply tube via a fluid element such as a hose. Relative to the exitopening, the supply container is arranged on a higher level, causing theimmersion medium to flow in the direction of the exit opening under theeffect of gravity. For control of the exiting quantity and of thefrequency of the immersion exit, a preferably controllable valve can beprovided in the fluid element.

[0018] By use of a pump or a supply container which optionally can alsobe connected to a pump, it is possible to supply immersion medium e.g.at fixedly predetermined points of time. Further, a continuous supply ofimmersion medium is possible.

[0019] In a preferred embodiment, a lens head of the objective issurrounded by a take-up means provided to receive excess immersionmedium. Such a take-up means is advantageous particularly in case of acontinuous supply of immersion medium. When the supply of immersionmedium is performed continuously or at predetermined time intervals, itis primarily due to the small gap between the exit lens of the objectiveand the outer surface of the sample support wall that the immersionmedium film provided between the exit lens of the objective and theouter surface of the sample support will not tear apart.

[0020] The take-up means is preferably provided as a take-up troughpreferably surrounding the whole lens head. This trough can have itsinner edge provided with a sealing lip or the like so that the take-uptrough can be placed around conventional lens heads.

[0021] For performing sequences of examinations—particularly longsequences—in high throughput screening systems, the take-up meanscomprises a drain opening provided to discharge liquid immersion mediumfrom the take-up means. Via the drain opening, the immersion medium canflow off, for instance. Preferably, the drain opening is provided with asuction means for suctional removal of excess immersion medium.

[0022] The gap provided between the exit lens of the objective and theouter surface of the sample support wall has a sizes in case of highthroughput screening methods, of less than 1000 μm, preferably less than500 μm and still more preferably less than 200 μm. With such a small gapwidth, a tearing-apart of the immersion medium in case of continuoussupply of immersion medium is avoided. Further, the medium will not tearas a result of a relative movement between the objective and the samplesupport, provided that the supply of a sufficient quantity of immersionmedium will be continued. Because of the capillary forces occurring insuch a small gap, it is also possible to arrange the above describedapparatus with the objective facing downwards.

[0023] The above disclosed apparatus is particularly suited for use inconfocal microscopes.

[0024] The invention further relates to a method for examination ofchemical and/or biological samples which here will particularly be anoptical examination method. According to the inventive method, a samplesupport receiving a sample is arranged in place relative to anobjective. The arrangement is performed in such a manner that a gap isformed between the outer surface of a sample support wall and an exitlens of the objective. If the objective is arranged below the samplesupport, the gap will thus extend between an underside of a samplesupport bottom and the exit lens. Afterwards, the immersion medium isautomatically introduced into the gap. This automatic supply makes theinventive method particularly suited for continuous examination methods.Especially in high throughput screening methods wherein a large numberof samples have to be measured at short time intervals, the inventivemethod will save considerable time and will yield examination results ofa considerably improved quality.

[0025] Preferably, the immersion medium is introduced into the gap usinga controllable supply device. The supply of the immersion medium can beperformed continuously, at regular intervals or exclusively as needed inthe given instance.

[0026] The refractive index of the immersion medium is preferably >1,particularly >1.3 to thus reach a numerical aperture of the objectiveof >1. This allows for the realization of an extremely small focus asrequired for a high resolution.

[0027] As an immersion medium, preferred use is made of water or oil.

[0028] The inventive method preferably includes a further step whereinexcess immersion medium is discharged.

[0029] Further, it is advantageous to introduce a cleansing liquid intothe gap prior to the supply of the immersion medium. In this manner, theouter lens of the objective and the sample carrier bottom can becleaned. As a cleansing liquid, e.g. alcohol can be used. Further, thepossibility exists to use different immersion media and, prior tointroduction of a new immersion medium, to clean the exit lens of theobjective as well as the outer surface of the sample support wall bysupplying a cleansing liquid. To accelerate such a process, it ispossible to provide a plurality of supply devices, wherein, forinstance, one supply device is used for supplying a first immersionmedium, a second supply device is used for supplying a cleansing liquid,and third supply device is used for supplying a second immersion medium.

[0030] Further, a feedback can be provided between the quantities ofimmersion medium which are supplied to the exit lens and those which aredischarged. Thereby, the quantity of the required immersion medium canbe monitored. For control of the quantity of immersion medium suppliedto the exit lens and the quantity of immersion medium discharged, avalve can be provided in the infeed line and/or the discharge line.

[0031] A preferred embodiment of the invention will be explained ingreater detail hereunder with reference to the accompanying drawings.

[0032]FIG. 1 shows a schematic side view of the apparatus of theinvention, and

[0033]FIG. 2 shows a schematic perspective view of the apparatusaccording to FIG. 1.

[0034] In the illustrated embodiment, the apparatus of the inventioncomprises an objective 10 with an exit lens 12 extending verticallyupwards. Arranged opposite the exit lens 12 is a sample support 14designed to receive a plurality of samples 16. The underside of samplesupport 14 is closed by a transparent sample support wall 18 which inthe illustrated embodiment is the sample support wall. By means of theobjective 10, examinations are performed on the individual samples 16.

[0035] The exit lens 12 of objective 10 which, for instance, can also bea protective glass cover or an auxiliary lens, is arranged at a distancefrom an outer surface of sample support 10. Thereby, a gap 22 is formedbetween the exit lens 12 and the outer surface 20 of the sample supportwall 18.

[0036] By means of a supply device 24, immersion medium provided in areservoir 26 is introduced into the gap 22. For this purpose, the supplydevice comprises a pump 28 connected to reservoir 26 via a hose 30.Connected to the pump 28 is a hose or tube 32 leading to a supply tube34. The end of supply tube 34 is formed similar to the tip of a pipetteand. has a flattened exit opening 36. To prevent the liquid column fromdropping, a valve 37 is provided.

[0037] For setting the distance of the exit opening 36 of supply tube 34relative to the exit lens 12, the tube 32 is held in a clamping means 38allowing the tube to be displaced in a longitudinal direction. Thus, theclamping means can be used for setting the position of exit opening 36relative to exit lens 12. To this effect, for instance, the clampingmeans 38 can also be arranged to be tilted about an axis extendingvertically to the plane of the drawing. In the illustrated embodiment,the exit opening 36 of supply tube 34 is arranged below the maximalpoint 40 of exit lens 12. Thus, exit opening 36 is arranged at adisplacement relative to the maximal point 40 of exit lens 12 in thedirection of objective 10.

[0038] The clamping means 38 is held by means of a bolt 42 to acollar-shaped take-up means 44 surrounding the objective 10. Bolt 42 canbe used for further adjustment of clamping means 38 and thus of exitopening 36 of supply tube 34. Take-up means 44 serves for taking upimmersion medium 46 provided between exit lens 12 and sample support 14if excess immersion medium is supplied via-supply device 24 to gap 22 orif the supplying is performed continuously.

[0039] The collar-shaped take-up means 44 comprises a discharge opening50 connected to a hose 48. Further, the hose 48 can have a device forsuctional removal connected thereto.

1. An apparatus for examination of chemical and/or biological samples,comprising a sample support for receiving the samples, an objective forobservation of the samples through a sample support wall, wherein a gapis formed between an outer surface of the sample support wall and anexit lens of the objectives, and a supply device for automatic supply ofan immersion medium between the outer surface of the sample support walland the exit lens of the objectives, characterized in that the supplydevice comprises a supply tube having its exit opening arranged close tothe exit lens.
 2. The apparatus according to claim 1, characterized inthat the supply tube is connected to the objectives.
 3. The apparatusaccording to claim 2, characterized in that the exit opening is arrangedat a distance of less than 15 mm, preferably less than 7 mm, from theexit lens.
 4. The apparatus according to any claim 1, characterized inthat the cross section of the exit opening is less than 1 mm²,preferably less than 0.8 mm².
 5. The apparatus according to claim 1,characterized in that the exit opening is arranged at a displacementrelative to a maximal point of the exit lens in the direction of theobjectives.
 6. The apparatus according to claim 5, characterized inthat, for supply of the immersion medium, a supply container is providedwhich is arranged at a different level relative to the exit opening. 7.The apparatus according to claim 5, characterized in that a controllablehose pump is provided for supply of the immersion medium.
 8. Theapparatus according to claim 1, characterized in that the supply tubehas a controllable valve connected thereto.
 9. The apparatus accordingto claim 1, characterized in that a lens head of the objective issurrounded by a take-up means for taking up excess immersion medium. 10.The apparatus according to claim 9, characterized in that the take-upmeans is formed as a take-up trough.
 11. The apparatus according toclaim 9, characterized in that the take-up means comprises a drainopening.
 12. The apparatus according to claim 11, characterized in thatthe drain opening is connected to a means for suctional removal.
 13. Theapparatus according to claim 1, characterized in that the gap has awidth of less than 1000 μm, preferably less than 500 μm and still morepreferably less than 200 μm.
 14. Use of the apparatus according to claim1, in a confocal microscope.
 15. A method for examination of chemicaland/or biological samples, comprising the steps of: arranging a samplesupport receiving the sample relative to an objective in such a mannerthat a gap is formed between an outer surface of a sample support walland an exit lens of the objectives, and subsequent automatic supply ofimmersion medium into the gap via a supply tube having its exit openingarranged close to the exit lens.
 16. The method according to claim 15,wherein the supply of the immersion medium is performed through acontrollable supply device.
 17. The method according to claim 15,wherein the immersion medium has a refractive index >1, preferably >1.3.18. The method according to claim 15, comprising the additional step of:discharging excess immersion medium.
 19. The method according to claim15, wherein, prior to the supply of the immersion medium, a cleansingliquid is supplied for cleaning the exit lens of the objective.